The stability and degradation pathway of recombinant human parathyroid hormone: deamidation of asparaginyl residue and peptide bond cleavage at aspartyl and asparaginyl residues.
OBJECTIVE
The stability of recombinant human parathyroid hormone (rhPTH) was examined under acidic to alkaline conditions; its degradation pathways were elucidated from resultant products.


METHODS
Degradation assay was performed in the pH range 2 to 10 at 40, 50 and 60 degrees C. The approximate molecular mass and pI values of the degradation products were estimated by electrophoresis. FAB-MS peptide mapping and amino acid composition analysis were used to determine these structures. The amount of each respective product was determined by HPLC.


RESULTS
At pH2, eight degradation products were found: 1-30rhPTH, 1-74rhPTH, 1-71rhPTH, 1-56rhPTH, 1-45rhPTH, 46-84rhPTH, 31-84rhPTH and Asp76-rhPTH; these were mainly as a consequence of peptide bond cleavage of the amide bond of Asp. At pH9, five products were found: isoAsp16-rhPTH, Asp16-rhPTH, Asp57-rhPTH, Asp76-rhPTH, 17-84rhPTH; the main degradation pathway was deamidation of Asn via a cyclic imide intermediate. Degradation products resulting from cleavage at Asp were increased in proportion to the extent that pH was lowered below 5. As pH was increased above 5, so were products resulting from deamidation of Asn. Correspondingly, levels of intact rhPTH were at a peak at pH5.


CONCLUSIONS
Degradation of rhPTH under acidic conditions predominantly occurs by cleavage at Asp, whereas, above pH5, deamidation of Asn is the more prominent, rhPTH is most stable at pH5.
Nabuchi
Y
Y
Fuji Gotemba Research Laboratories, Chugai Pharmaceutical Co., Ltd., Shizuoka, Japan.
Fujiwara
E
E
Kuboniwa
H
H
Asoh
Y
Y
Ushio
H
H
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D001216:Asparagine / Q000737:chemistry*; D001224:Aspartic Acid / Q000737:chemistry*; D004355:Drug Stability; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D010281:Parathyroid Hormone / Q000737:chemistry* / Q000378:metabolism*; D010446:Peptide Fragments / Q000737:chemistry / Q000302:isolation & purification; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D012697:Serine Endopeptidases; D016339:Spectrometry, Mass, Fast Atom Bombardment; D013696:Temperature; D014357:Trypsin
D000596:Amino Acids; D010281:Parathyroid Hormone; D010446:Peptide Fragments; D011994:Recombinant Proteins; D001224:Aspartic Acid; D001216:Asparagine; D012697:Serine Endopeptidases; C028344:glutamyl endopeptidase; D014357:Trypsin